Molecular Cloning Of Ubiquitin Specific Protease 12

Objective: Protein degradation regulated by ubiquitination in cells controls a number of biological processes, including cell cycle progression, DNA-repair, transcriptional regulation, immune response, and signal transduction. The conjugation of ubiquitin to proteins is catalyzed by the sequential actions of ubiquitin activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin-protein ligases (E3). The removal of the ubiquitin from proteins, negative regulators of protein ubiquitination, is catalyzed by deubiquitinating enzymes (DUBs). The human genome encodes 95 Dubs in five distinct subfamilies based on their sequence similarities and likely mechanisms of action: ubiquitin specific proteases (USPs), ubiquitin C-terminal hydrolases (UCHs), otubain domain-containing proteins (OTUs), Machado-Joseph domain (Josephin domain)-containing proteins (MJD) and JAMM zinc metalloproteinase domain-containing proteins (JAMM).The largest and most diverse of these subfamilies is the ubiquitin specific protease. USPs are different in length and structure but contain several highly conserved Cys, His and Asp domains in their amino acid sequence. The current study found that USPs regulate various biological and pathological processes such as stabilization of the p53 tumor suppressor, regulation of cellular growth pathways, anti-apoptotic effect, modulation of developmental processes, or inhibition of gene silencing.In this study, we have isolated and characterized a novel USP, usp12, from rat brain and human A549 cell. We also analyze the tissue distribution of the rat usp12 and perform an analysis of their enzymatic activity. Both the rat USP12 and human USP12 have deubiquitinating enzyme activity, but the SNP mutant form of human USP12, containing H173D, failed to cleave the model substrate. This result indicates that the SNP may play an important role in human. Methods:(1) Cloning of rat usp12. Total RNA was isolated from the rat brain using TRNzol. First strand cDNA was synthesized from total RNA using Quant Reverse Transcriptase and PCR was performed with the usp12-specific primers. The PCR amplified fragment with a predicted size was subcloned into pGEM-T easy vector and introduced into E. coli DH5α. After selected by digestion with BamH I and Sal I, the plasmid containing a predicted fragment was sequenced.(2) Tissue distribution of the rat usp12 was analyzed by Quantitative real-time PCR. RNA isolation was performed using TRNzol from a variety of tissues including brain, heart, kidney, liver, lung, skeletal muscle, spleen, and testis. Real-time PCR analysis was performed using rat usp12-specific primers and rat GAPDH-specific primers with Quant one step qRT-PCR (SYBR Green I) kit. The results were analyzed based on threshold cycle (Ct) values. Expression differences were determined by raising 2 to the -ΔΔCt power.(3) Expression of the rat USP12 fusion protein. After digestion with BamH I and Sal I, the rat usp12 fragment was inserted into pGEX-6p-1, downstream of the Glutathione S-transferase (GST) coding element, and then transformed into E. coli DH5αcells. After 4 h induction with the IPTG, the total, supernatants and pellets of lysed cell were analyzed by 10% SDS-PAGE.(4) Deubiquitinating enzyme activity assay. The expression vectors pGEX-6p-1, pRB-UBP2, pGEX-rusp12 and pGEX-rusp12(C48S) were co-transformed into E. coli BL21 competent cells with pAC-ub-met-β-gal. After induction with IPTG, protein extracts were analyzed by 7% SDS-PAGE and Western blot with a mouse anti-β-gal antibody.(5) Cloning of human usp12. Total RNA was extracted from A549 cell using TRNzol. PCR were performed using the usp12-specific primers. After inserted into pGEX-6p-1, site-directed mutagenesis on the histidine at position 173 was carried out with Pfu DNA polymerase. Deubiquitinating enzyme activity assay was performed with Ub-Met-β-gal.Results: (1) Rat USP12 consists of 1113 bp and encodes 370 amino acids with a molecular weight of approximately 43 KDa. Rat USP12 contains the highly conserved Cys, His, and Asp domains characteristic of the ubiquitin specific proteases.(2) Expression of the usp12 gene was detected in all rat tissues tested, with particularly strong expression in the spleen and kidney, and weaker expression was found in the tissues of heart and skeleton muscle.(3) After induction with IPTG, The USP12 fusion protein, GST-rUSP12, expressed in the E. coli DH5αwith a molecular weight of approximately 70 KDa as determined in SDS-PAGE analysis.(4) The Deubiquitinating enzyme activity assay showed that rat USP12 has deubiquitinating enzyme activity as it cleaves the ubiquitin from Ub-Met-β-gal.(5) Human USP12 consists of 1113 bp and encodes 370 amino acids with a molecular weight of approximately 43 KDa. Human USP12 have deubiquitinating enzyme activity, but the SNP mutant form of human USP12, containing H173D, failed to cleave the model substrate.Conclusions:(1) We have cloned a novel ubiquitin specific protease, USP12, from rat brain.(2) USP12 was expressed in various rat tissues, with particularly strong expression in the spleen and kidney.(3) The USP12 fusion protein, GST-rUSP12, expressed in the E. coli DH5α.(4) Rat USP12 has deubiquitinating enzymatic activity.(5) We also isolated human USP12 from A549 cell. Human USP12 have deubiquitinating enzyme activity, but the SNP mutant form of human USP12, containing H173D, showed no activity.